The melanocortin system in the mammalian forebrain

Leptin Increases Hypothalamic Pro-opiomelanocortin mrna Expression in the Rostral Arcuate Nucleus Michael W. Schwartz, Randy J. Seeley, Stephen C. Woods, David S. Weigle, L. Arthur Campfield, Paul Burn, and Denis G. Baskin Melanocortins are peptides, cleaved from the pro-opiomelanocortin (POMC) precursor, that act in the brain to reduce food intake and are potential mediators of leptin action. In the forebrain, melanocortins are derived from POMC-containing neurons of the hypothalamic arcuate nucleus. To test the hypothesis that these POMC neurons are regulated by leptin, we used in situ hybridization to determine whether reduced leptin signaling (as occurs in fasting), genetic leptin deficiency (in obese ob/ob mice), or genetic leptin resistance (in obese db/db mice) lower expression of. We further hypothesized that leptin administration would raise hypothalamic levels in leptin-deficient animals, but not in mice with defective leptin receptors. In wild-type mice (n = 12), fasting for 48 h lowered levels in the rostral arcuate nucleus by 53%, relative to values in fed controls (n = 8; P < 0.001). Similarly, arcuate nucleus levels were reduced by 46 and 70% in genetically obese ob/ob (ji = 6) and db/db mice (n = 6), respectively, as compared with wild-type mice (n = 5) (P < 0.01 for both comparisons). Five daily intraperitoneal injections of recombinant murine leptin (150 ug) raised levels of in the rostral arcuate nucleus of ob/ob mice (#i = 8) by 73% over saline-treated ob/ob control values (re = 8; P < 0.01), but was without effect in db/db mice (re = 6). In normal rats, two injections of a low dose of leptin (3.5 ug) into the third cerebral ventricle (re = 15) during a 40-h period of fasting also increased POMC mrna levels in the rostral arcuate nucleus to values 39% greater than those in vehicle-treated controls (re = 14; P = 0.02). We conclude that reduced central nervous system leptin signaling owing to fasting or to genetic defects in leptin or its receptor lower levels in the rostral arcuate nucleus. The finding that leptin reverses this effect in ob/ob, but not db/db, mice suggests that leptin stimulates arcuate nucleus POMC gene expression via a pathway involving leptin receptors. These findings support the hypothesis that leptin sig- From the Departments of Medicine (M.W.S., D.S.W., D.G.B.), Psychology (R.J.S., S.C.W.), and Biological Structure (D.G.B.), University of Washington, Pugent Sound VA Health Care System, and Harborview Medical Center, Seattle, Washington; and the Department of Metabolic Diseases (L.A.C., P.B.), Hoffmann-LaRoche, Nutley, New Jersey. Address correspondence and reprint requests to Michael W. Schwartz, MD, Metabolism (151), Puget Sound VA Health Care System, 1660 S. Columbian Way, Seattle, WA 98108. E-mail: mschwart@u.washington.edu. Received for publication 17 September 1997 and accepted in revised form 8 October 1997. a-msh, a-melanocyte-stimulating hormone; ANOVA, analysis of variance; CRH, corticotropin-releasing hormone; CSF, cerebrospinal fluid; ISH, in situ hybridization; MC4-R, melanocortin 4-receptor; NPY, neuropeptide Y; POMC, pro-opiomelanocortin. naling in the brain involves activation of the hypothalamic melanocortin system. Diabetes 46:2119-2123, 1997 The melanocortin system in the mammalian forebrain comprises peptides, such as a-melanocyte-stimulating hormone (a-msh), that are cleaved from the POMC precursor polypeptide synthesized by neurons of the hypothalamic arcuate nucleus (1). After their release from axon terminals, these peptides bind to and activate neuronal melanocortin receptors (2). The recent finding that agonists of the melanocortin 4-receptor (MC4-R) subtype cause anorexia and weight loss after intracerebroventricular administration (3) focused attention on the possibility that the melanocortin system plays a major role in energy homeostasis. The observation that severe obesity results from targeted deletion ("knockout") of the MC4- R provides compelling evidence that melanocortin signaling is necessary for normal control of adipose stores (4). The adipocyte hormone, leptin, is critically involved in body weight regulation. like agonists of the MC4-R, leptin also causes sustained anorexia and weight loss after intracerebroventricular administration (5,14). Since POMC neurons in the arcuate nucleus express leptin receptors (6), we hypothesized that leptin action in the brain involves stimulation of the melanocortin system. Consistent with this hypothesis, it has been reported that the ability of intracerebroventricular leptin to reduce food intake and body weight and to induce c-fos expression in the hypothalamic paraventricular nucleus of rats is blocked by intracerebroventricular pretreatment with the MC4-R antagonist, SHU9119 (7). These actions of leptin in the brain therefore appear to require signaling at MC4-Rs. On the basis of these observations, we hypothesized that conditions associated with either reduced leptin levels (e.g., during fasting or in leptin-deficient ob/ob mice) or defective leptin receptors (e.g., db/db mice) should lower POMC gene expression in neurons of the arcuate nucleus, and that this effect should be reversed by leptin administration to animals that are able to respond to the leptin signal. On the basis of the finding that fasting lowers levels in the rostral portion of the arcuate nucleus of rats (8), we used in situ hybridization (ISH) to focus our studies on this specific brain area. RESEARCH DESIGN AND METHODS Study animals and procedures. All animals were maintained on a 12-h day:night schedule with ad libitum access to standard laboratory diet and water DIABETES, VOL. 46, DECEMBER 1997 2119

LEPTIN STIMULATES POMC GENE EXPRESSION unless otherwise specified. All procedures were in accordance with institutional guidelines for animal care of the University of Washington. Experiment 1: Effect of fasting and mutations ofleptin or its receptor. Wild-type male mice weighing 27.2 ± 1.0 g were either fed ad libitum (n = 8) or had food removed for 48 h before being killed (n = 12). After euthanasia by inhalation of CO 2, mice were decapitated and brains rapidly removed, frozen in a bed of powdered dry ice, and stored at -70 C. In a separate study of mice with continuous ad libitum access to standard laboratory diet, male C57B1/6J ob/ob (n = 6), C57B1/Ks db/db mice (n = 6), and wild-type control mice (n = 5) were killed and brains were collected as above. Experiment 2: Effect of systemic leptin in leptin-deficient and leptin-resistant mice. Male ob/ob and db/db mice were treated for 5 days with daily intraperitoneal injections of either saline or recombinant murine leptin (150 ug/injection). In 06/06 mice, pair-feeding was performed in a separate group of saline-treated animals by assigning each pair-fed ob/ob mouse to a partner in the leptin treatment group. The amount of standard diet provided to each pair-fed animal on each treatment day was equal to the measured amount of standard diet consumed by its leptin-treated partner during the previous 24-h period. Mice were anesthetized by ether inhalation and killed as described for experiment 1. Measures of food intake, body weight, and hypothalamic neuropeptide Y (NPY) and corticotropinreleasing hormone (CRH) mrna levels from this experiment were reported previously (9). Experiment 3: Effect ofintracerebroventricular leptin in food-deprived rats. Male Long-Evans rats (350-450 g) obtained from the breeding colony maintained by the Department of Psychology at the University of Washington were housed individually in wire-mesh hanging cages in a temperature-controlled vivarium on a 12-h light:dark cycle. Rats were anesthetized with Equithesin (3.4 ml/kg) and a cannula inserted stereotaxically into the third cerebral ventricle as previously described (5,10). Recombinant human leptin (Hoffmann-LaRoche, Nutley, NJ), dissolved in artificial cerebrospinal fluid (CSF) (1.0 ug/ul) or its vehicle (artificial CSF with a protein content matched to that in the leptin preparation), was injected intracerebroventricularly in a volume of 3.5 ul over 1 min in lightly restrained, conscious rats at both the onset and 16 h before the conclusion of a 40-h fast. After decapitation, brains were handled as described for experiment 1. Body weight and hypothalamic NPY and CRH mrna levels from this experiment were reported previously (10). In situ hybridization. Brains removed immediately after decapitation were frozen on dry ice and sectioned at 14 um with a cryostat. Slides for ISH to POMC mrna were selected from the region of the arcuate nucleus rostral to the ventromedial hypothalamic nucleus by one of the authors blinded to treatment group to ensure anatomic equivalence of hypothalamic sections among animals. ISH was performed using ^P-labeled riboprobe, complementary to both mouse and rat (a generous gift of Dr. Robert Steiner), and mrna hybridization quantitated by computer densitometry of ISH autoradiograms as described previously (11,12). The average total hybridization area (pixels) and pixel density (calibrated to microcuries per gram per pixel) were recorded from 6 to 8 sections for each brain, and background density was subtracted, producing a single value for each brain area on each section. These data are presented as the product of hybridization area x density. Statistical analysis. Comparisons between group mean values of were performed using either an unpaired Student's t test (for two-group comparisons) or one-way analysis of variance (ANOVA) and Fisher's test for multiple comparisons (for comparisons of three groups). The null hypotheses of no difference between groups were rejected atp < 0.05. RESULTS Experiment 1. In wild-type male mice, a period of 48 h of fasting (n = 8) decreased body weight by 27% (from 27.2 ± 1.0 to 19.5 ± 0.5 g) and reduced hybridization in the rostral arcuate nucleus by 53%, as compared with ad libitum fed controls (n = 12; P < 0.01) (Fig. L4). This finding was reproduced in a second group of normal mice, in which fasting for 48 h (n = 4) reduced in the rostral arcuate nucleus by 41 ± 8%, relative to fed controls (n = 6; P < 0.05), but did not alter levels in the midregion of the arcuate nucleus (108 ± 13 vs. 100 ± 12% for fasted vs. fed; NS). Therefore, we limited subsequent analysis of levels to the rostral arcuate nucleus. In this brain area, POMC mrna levels were also reduced by 46 and 70% in genetically obese ob/ob and db/db mice, respectively, relative to wild-type controls (P < 0.01 for both comparisons; Fig. IB). In mice, therefore, fasting, genetic leptin deficiency and genetic leptin resistance were each associated with a marked decrease of in the rostral arcuate nucleus. Experiment 2. Five daily intraperitoneal injections ofleptin lowered body weight by 4.1% (P < 0.05) (9) and increased hybridization in the rostral arcuate nucleus of ob/ob mice by 73% (P < 0.05), as compared with saline-treated ob/ob mice fed ad libitum (Fig. 2). Whereas pair-feeding of saline-treated ob/ob mice to the intake of those mice receiving leptin produced a comparable degree of weight loss (-3.4%; NS vs. leptin-treated ob/ob mice) (9), this intervention did not significantly affect levels in this brain area. Leptin administration to db/db mice also was without effect on levels in the rostral arcuate nucleus (Fig-2). Experiment 3. Two injections of leptin (3.5 ug) into the third cerebral ventricle of rats during a 40-h period of food deprivation resulted in a significant increase of 39% in POMC mrna levels in the rostral arcuate nucleus, as compared with intracerebroventricular vehicle-treated controls (Fig. 3). Thus, leptin administered directly into the brain of fasted, genetically normal rats increased levels in the same brain area that responded to leptin in ob/ob mice. DISCUSSION Recent studies implicate the melanocortin system in the brain as an important pathway for the normal control of energy homeostasis (3,4) that may mediate at least some central nervous system responses to leptin (7). These observations led us to hypothesize that expression of the POMC gene by arcuate nucleus neurons (the source of POMC in the forebrain) should be stimulated by leptin, and that reduced leptin signaling owing either to genetic or to acquired causes should lower levels in this brain area. Our results provide direct support for this hypothesis. We found that conditions associated with either relative (e.g., fasting) or absolute leptin deficiency (e.g., obese ob/ob mice) were associated with levels in the rostral arcuate nucleus that were approximately one-half of control values, as determined by ISH. The hypothesis that this response resulted from reduced leptin signaling in the brain is supported by several additional findings. First, genetic obesity owing to mutation of the leptin receptor gene in db/db mice was also associated with a marked decrease of in this brain area. Since these animals cannot respond to leptin (13-15), our findings suggest that reduced leptin receptor signaling lowers POMC gene expression by neurons in the rostral arcuate nucleus in a manner comparable to that seen in genetic leptin deficiency. Direct support for this conclusion was provided by measuring the effect of systemic leptin administration on POMC gene expression in the hypothalamus of ob/ob and db/db mice. As predicted by our hypothesis, leptin treatment for 5 days increased POMC mrna expression by 73% in the rostral arcuate nucleus of ob/ob mice. However, since leptin had no effect on arcuate nucleus in db/db mice, it appears that normal leptin receptors are required for this response. In addition, pairfeeding of saline-treated ob/ob mice to the food intake of leptin-treated animals did not increase expression despite a comparable decrease in body weight (9). This finding indicates that leptin-induced weight loss is not responsible for its effect to increase POMC gene expression in the hypothalamus of ob/ob mice. 2120 DIABETES, VOL. 46, DECEMBER 1997

M.W. SCHWARTZ AND ASSOCIATES 120" A 200i To investigate whether the effect of leptin on POMC gene expression occurs directly within the brain and whether it can be elicited in genetically normal animals, we measured in the rostral arcuate nucleus of fasted rats receiving either a low dose of leptin (two doses of 3.5 ug during a 40-h fast) or its vehicle iryected directly into the third cerebral ventricle. Our finding that intracerebroventricular leptin increased levels in this brain area suggests that the previous report of reduced in the arcuate nucleus of fasted rats (8) was attributable, at least in part, to reduced leptin signaling. Leptin, therefore, appears to be a major regulator of POMC gene expression in the rostral arcuate nucleus of both mice and rats. Our finding that fasting did not alter levels in the midregion of the arcuate nucleus of mice is consistent with an earlier study in rats (8). This observation raises the possibility that a subpopulation of POMC neurons that are sensitive to leptin may be highly localized to the rostral aspect of the arcuate nucleus. This possibility is consistent with functional heterogeneity described for other hypothal- 150-100' 20' fed (%fed value) fasted saline pairfed leptin ~ saline leptin ob/ob db/db (% ob/ob saline) 120i B 100-80- 60-40- 100-80- 60 40 201 WT ob/ob db/db (% WT) FIG. 1. Levels of in the rostral arcuate nucleus determined by in situ hybridization. A: effect of fasting for 48 h (n = 12) as compared with ad libitum feeding (n = 8) in male wild-type mice. *P = 0.0002 by unpaired two-tailed Students' t test. B: comparison of ob/ob mice (n = 6) and db/db mice (n = 6) to wild-type controls (ra = 5). *P < 0.05 vs. wild-type by one-way ANOVA. FIG. 2. levels in the rostral arcuate nucleus of ob/ob and db/db mice receiving daily intraperitoneal injections of either 150 ug of recombinant murine leptin (leptin; n = 8) or saline for 5 days. Saline-treated mice were either fed ad libitum (saline; n = 8) or pairfed to the food intake of leptin-treated mice (pair-fed; n = 8). *P < 0.05 vs. saline-treated ob/ob mice. amic neuropeptide systems in this brain area. For example, fasting increases NPY mrna in the midregion and rostral portions of the arcuate nucleus, but has no detectable effect in its caudal portion (16). These considerations stress the importance of tools that permit detection of highly localized changes in hypothalamic gene expression, such as ISH, and their application to the relevant anatomical region. Regional variability in leptin responsiveness of POMC neurons may explain the normal level recently reported in the midregion of the arcuate nucleus of ob/ob mice (17). While our results do not establish hypothalamic POMC neurons as downstream mediators of leptin action in the control of food intake and energy balance, they strengthen a growing database in support of this concept. Leptin receptor mrna is concentrated in the arcuate nucleus (10,18) and was recently found to be highly colocalized with POMC mrna in neurons in this brain area (6), suggesting that POMC neurons are a target of leptin action in the brain. In addition, two mouse models of genetic obesity implicate defective melanocortin signaling in the brain as a cause of weight gain associated with hyperleptinemia, suggesting a leptin-resistant state. The A y agouti mouse is characterized by ubiquitous overexpression of agouti, an endogenous antagonist of melanocortin receptors normally found only in hair follicles. This genetic defect results in a syndrome of hyperphagic obesity, hyperleptinemia, and yellow coat color. The finding that "knockout" of MC4-Rs by targeted disruption also causes obesity and hyperleptinemia (4) suggests that impaired melanocortin signaling, resulting either from antagonism of melanocortin receptors by agouti or from MC4-R deficiency, causes a syndrome in which excessive weight gain occurs despite elevated leptin levels, consistent with leptin resistance (19). This concept is also consistent with recent evidence that pharmacological blockade of MC4-Rs impairs leptin's ability to cause anorexia and weight loss (7). Thus, activation of hypothalamic POMC-containing neurons may be one mech- DIABETES, VOL. 46, DECEMBER 1997 2121

LEPTIN STIMULATES POMC GENE EXPRESSION We thank Daniel Porte, Jr., for helpful comments on the design of these studies and on this manuscript and also Jay Erickson and Richard Palmiter for their assistance. Technical assistance was provided by Vicki Hoagland and Zoe Jonak. Some of the tissue and materials for the experiments involving the ob/ob and db/db mice were provided by Zymogenetics and involved the assistance of Joseph Kuijper, Tom Bukowski, Janet Kramer, and Margaret Fallon. The riboprobe for was generously provided by Robert Steiner. icv vehicle icv leptin (% icv vehicle) FIG. 3. Effect of intracerebroventricular leptin administration on in the rostral arcuate nucleus of food-deprived rats. Leptin (n = 15) or its vehicle (n = 14) was administered into the third cerebral ventricle at the beginning and after 24 h of a 40-h period of fasting. *P = 0.01 vs. vehicle. anism by which leptin lowers body weight, and disruption of this cascade of events may result in excess weight gain. This hypothesis takes on greater significance in light of the report that hyperleptinemia and obesity in humans are linked to a locus on chromosome 2 near the POMC gene (20). The possibility that a mutation in the POMC gene contributes to leptin resistance and obesity in humans is consistent with our findings. In addition to its anorexic and weight-reducing effects, leptin influences a variety of neuroendocrine and autonomic responses (6,21-23). Further study is therefore required before conclusions can be drawn regarding the role of the hypothalamic melanocortin pathway as a mediator of leptin action in systems outside those regulating energy homeostasis. Activation of the arcuate nucleus pathway containing NPY (a potent stimulant of food intake) is implicated in the hypothalamic response to genetic or acquired leptin deficiency (9,24-27). However, NPY is unlikely to be a major mediator of leptin-induced anorexia, since NPY-deficient mice exhibit a robust anorexic response to leptin (28). The response to leptin deficiency may therefore involve a cascade of events that are not simply the reverse of the response to leptin (7,25,29). During leptin deficiency, we hypothesize that activation of NPY neurons plays a major role, and perhaps in concert with inhibition of melanocortin pathways, promotes increased food intake and weight gain. The anorexic response to leptin, however, may be mediated predominantly by activation of hypothalamic circuits that reduce food intake and increase energy expenditure, such as pathways containing POMC and CRH (10,25,29). An improved understanding of the hypothalamic mechanisms mediating leptin signaling in the brain may eventually lead to the development of effective new treatments of human obesity and related disorders. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health, the Diabetes Endocrinology Research Center, the Clinical Nutrition Research Unit at the University of Washington, and the VA Merit Review program. REFERENCES 1. Eskay RL, Giraud P, Oliver C, Brown MJ: Distribution of a-melanocyte-stimulating hormone in the rat brain: evidence thata-msh-containing cells in the arcuate region send projections to extrahypothalamic areas. Brain Res 178:55-67, 1979 2. Mountjoy K, Mortrud M, Low M, Simerly R, Cone R: Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain. Mol Endocrinol 8:1298-1308,1994 3. 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